The invention relates to an apparatus, the application or use of the apparatus and separation media for separating particles by means of a free flow electrophoresis technique in accordance with the generic part given in the independent claims.
A major difficulty in diagnostic practice and research involves precisely defined sample preparations. However, the technologies in common use today, such as centrifugation, filtration, magnetic separation and FACS (Fluorescence Activated Cell Sorting) have various disadvantages, such as restricted specificity, low throughput rates, limited purification ability or also expensive apparatus for isolating and purifying cells, organelles or biomolecules (e.g. protein complexes). The most important applications for purifying cells, organelles and biomolecules (e.g. protein complexes) are found in the fields of Biopharmacy, Biotechnology, Environmental Analysis, Food Technology and Medicine. The applied techniques are either preparative (Biopharmacy and Biotechnology) or analytic (Environmental Analysis, Food Technology and Medicine) in scope.
Free-flow electrophoresis (FFE) is one of the most promising technologies for separating all possible particles [cf. Krivanova L. & Bocek P. (1998), “Continuous free-flow electrophoresis”, Electrophoresis 19: 1064–1074]. In the field of Proteomics FFE is the technology of choice for the defined preseparation of complex protein samples in terms of their varying pI values (degree of ionization). Using FFE, the cells are separated on the basis of the electrophoretic mobility of the cells. The corresponding principles have already been characterized in depth [cf. e.g. Bondy B., Bauer J., Seuffert I. and Weber G. (1995), “Sodium chloride in separation medium enhances cell compatibility of free-flow electrophoresis”, Electrophoresis 16: 92–97], whereas FFE has received little recognition as most cell types differ only minimally in terms of their surface charge, making the separation of these cell types difficult. One of the first refinements was the introduction of immuno-FFE which allowed specific antibodies to be bound to specific surface epitopes of the cells to be separated, making it possible to modify the electrophoretic mobility of these cells in FFE by modifying the net charge of the cell surface [cf. e.g. Hansen E. and Hannig K. (1982), “Antigen-specific electrophoretic cell separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies”, J. Immunol. Methods 11, 51: 197–208].
An FFE apparatus of the same generic type is described in the international patent application PCT/EP01/14408. It concerns an electrophoresis apparatus with a separation chamber through which the separation medium flows and which is a space defined by a floor and a cover and spacers separating these two from each other. In addition, this FFE apparatus encompasses a pump for supplying the separation medium which enters the separation chamber via medium feed lines and leaves the chamber via outlets. The FFE apparatus also includes electrodes for applying an electric field within the separation medium and sample injection points for adding the mixture of particles or analytes and fractionation points for removing the particles separated by FFE in the separation medium. These separated particles can be used for analytic purposes or for further preparative processing. In order to exert an influence upon the separation medium flow profile provision can be made for two or more separate dosage pump feed lines to add medium which are connected to the separation chamber in the vicinity of the fractionation points in the vicinity of the electrodes.
The patent U.S. Pat. No. 5,948,231 discloses compositions, methods, and apparatus for performing ultrafast binding assays by capillary electrophoresis or other electro-separation techniques, such as Free Flow Electrophoresis.